Small populations of stem cells sustain the limbal-corneal and the conjunctival lineages. In the limbal-corneal system, where stem cells localize preferentially to the limbus, total or zonal damage produces epithelial limbal stem cell deficiency syndromes. In recent years, a variety of surgical stem cell transplant methodologies have been applied to reestablish the limbal stem cell niche for vision restoration. The long term goals of our studies are a) understand the functional nature of ocular surface stem cells, in particular those of the limbus; and b) apply this critical knowledge for the optimization of ex vivo limbal cell population expansion to improve surface reconstructive procedures based on transplantation of stem cell-rich cell populations. During the initial funding period of this grant we characterized small stem cell cohorts known as side populations (SPs) from both conjunctival and limbal epithelia. Our studies showed that these cells derive from cells that have been in the slow cycling state in vivo and to display several other features recognized for stem cells in vitro. Based on finding during this period we now propose, in Specific Aim1 to test the hypothesis that cell cohorts expressing CD62E or excluding mitotrakcker deep red constitute additional component of an heterogeneous stem/precursor limbal and conjunctival epithelial cell population and in Specific Aim2 to test the hypothesis that observed overexpressions of several genes capable of affecting gene transcription globally and usually referred as master or cell fate genes (PAX6, MEIS1, SIX1, MSX1, HES1 ID1, the MXD1/MAX system controlling MYC activity) contribute to cell stemness in the limbal epithelium. The knowledge acquired in the comparative characterization of the cell cohorts associated with the stem/precursor cell compartment in Aim 1, combined with the identification of gene expressions and signal pathways that are associated with preservation or loss of the stem/precursor cell state in Specific Aim 2, will provide the cogent basis to improve strategies for either maximization of stem/precursor cell yields during limbal cells expansion in vitro, or alternatively, facilitate stem cell rescue from generic proliferative (basal) epithelial cells through reversion of phenotype for the benefit of reconstructive ocular surface procedures. Specific aim 3 seeks to apply the knowledge and expertise acquired in the two previous specific aims for the genetic and/pharmacological manipulation of freshly isolated limbal epithelial cells to attain effective expansion of these cells while maintaining their capacity for corneal epithelial function recovery in vivo.